Many solid phase resins and handles for peptide synthesis are available in the art, as is described in Fields and Noble, 1990, Int. J. Pept. Protein Res. 35:161-214. To be useful, handles must be stable to the reaction conditions of peptide synthesis, but when the synthesis is complete, the handle needs to allow for cleavage of the peptide from the solid support.
Less common, but no less useful, are selectively cleavable linkers. These linkers can act as handles; alternatively, the linker can be coupled to another handle. The useful feature of certain of these linkers is that they can be cleaved under much milder conditions than are associated with handles, e.g., under mildly basic or acidic conditions, rather than highly acidic conditions associated with peptide cleavage, e.g., in hydrogen fluoride (HF) or trifluoroacetic acid (TFA).
Ultraviolet light sensitive linkers, such as ONb (see Barany and Albericio, 1985, J. Am. Chem. Soc. 107:4936-4942) have been used. Other cleavable linkers require hydrogenolysis or photolysis. Examples of other photosensitive (photocleavable) linkers are found in Wang (1976, J. Org. Chem. 41:32-58), Hammer et al. (1990, Int. J. Pept. Protein Res. 36:31-45), and Kreib-Cordonier et al. (1990, in Peptides-Chemistry, Structure and Biology, Rivier and Marshall, eds., pp. 895-897).
Landen (1977, Methods Enzym. 47:145-149) used aqueous formic acid to cleave Asp-Pro bonds; this approach has been used to characterize T-cell determinants in conjunction with the Geysen pin synthesis method (Van der Zee et al., 1989, Eur. J. Immunol. 191:43-47). However, treatment of peptides with formic acid is undesirable, especially if the treated peptides are to be used in a biological assay.
Other potential linker groups cleavable under basic conditions include those based on p-(hydroxymethyl)benzoic acid (Atherton et al., 1981, J. Chem. Soc. Perkin I:538-546) and hydroxyacetic acid (Baleaux et al., 1986, Int. J. Pept. Protein Res. 28:22-28). Geysen et al. (1990, J. Immunol. Methods 134:23-33) reported peptide cleavage by nucleophilic attack on a carboxy-terminal ester of proline, giving rise to a two-ring derivative of diketopiperazine. However, the "diketopiperazine" method described by Geysen produces peptides which carry a C-terminal diketopiperazine moiety (Bray et al., 1991, J. Org. Chem. 56:6659-6671). This is a disadvantage, because the heterocyclic moiety can affect binding activity or biological activity of the released peptide. International Patent Publication WO 90/09395 (Geysen, published Aug. 23, 1990) suggests orienting a dipeptide cleavable linker so that the heterocycle remains on the solid support.
Moreover, recently techniques have been developed that make possible the preparation of libraries of 10.sup.6 to 10.sup.7 or more peptides attached to solid phase supports, in which each particle of solid phase support contains a single peptide species (International Patent Publication No. W092/00091, published Jan. 9, 1992). These libraries are used advantageously when the peptide species can be sequentially cleaved in substantially equimolar amount from the solid phase support, so that each peptide species can be tested in more than one assay for its activity, or peptides can be tested in mixtures, followed by separation of all the resin particles from a positive mixture for further testing. These libraries also provide for an equimolar amount of each peptide to remain on the solid phase support for sequencing those peptides that, when released from the solid support, exhibit the activity of interest.
Thus there is a need in the art for a releasable linker that yields a peptide of substantially unaltered structure, and in particular, which does not have a diketopiperazine moiety attached. Moreover, there is a need for release of peptides to a solution compatible with biological tests.
Furthermore, there is a need in the art for multiply cleavable linkers, in which cleavage of each linker is independent of cleavage of the others, i.e., orthogonal to cleavage of the others, thus providing for sequential cleavage of the same or different peptide species from a solid support in equimolar ratios.
Citation of any reference in this application is not to be deemed an admission that such reference is available as "prior art."